Interactions of hemoglobin (Hb) with small diffusible ligands, such as O.sub.2, CO.sub.2 and NO, are known to occur at its metal centers and amino termini. The O.sub.2 /CO.sub.2 delivery functionalities, which arise in the lung and systemic microvasculature, are allosterically controlled. Such responsiveness to the environment is not known to apply in the case of NO. Specifically, it is thought that Hb(Fe) is involved in limiting NO's sphere of action (Lancaster, J. R., Proc. Natl. Acad. Sci. USA, 91:8137-8141 (1994); Wood and Garthwaite, J. Neuropharmacol., 33:1235-1244 (1994)), but that NO does not modify the functional properties of Hb to any physiologically significant degree. Kinetic modeling based on this assumption, however, predicts that the vast majority of free NO in the vasculature should be scavenged by Hb (Lancaster 1994). Accordingly, the steady-state level of NO may fall below the K.sub.m for target enzymes such as guanylate cyclase (Lancaster 1994), if not in the unperturbed organism, then with oxidant stress such as that found in atherosclerosis. These considerations raise the fundamental question of how NO exerts its biological activity.
One answer to this paradox may be found in the propensity of nitric oxide to form S-nitrosothiols (RSNOs) (Gaston, B. et al., Proc. Natl. Acad. Sci. USA, 90:10957-10961 (1993)), which retain NO-like vasorelaxant activity (Stamler, J. S., et al., Proc. Natl. Acad. Sci, USA, 89:444-448 (1992)), but which are not subject to the diffusional constraints imposed by the high concentration of Hb in the blood. In particular, the NO group of RSNOs possesses nitrosonium (NO.sup.+) character that distinguishes it from NO itself. Indeed, it is increasingly appreciated that RSNO's have the capacity to elicit certain functions that NO is incapable of (DeGroote, M. A. et al., Proc. Natl. Acad. Sci. USA, 92:6399-6403 (1995); Stamler, J. S., Cell, 78:931-936 (1994)). Moreover, consideration has been given to the possibility that -SNO groups in proteins may serve a signaling function, perhaps analagous to phosphorylation (Stamler, J. S. et al., Proc. Natl. Acad. Sci. USA, 89:444-448 (1992); Stamler, J. S. Cell, 78:931-926 (1994)). Although S-nitrosylation of proteins can regulate protein function (Stamler, J. S. et al., Proc. Natl. Acad. Sci. USA, 89:444-448 (1992); Stamler, J. S., Cell, 78:931-936 (1994)), the identification of S-nitrosoproteins within cells--the sine qua non of a regulatory posttranslational modification--has heretofore not been demonstrated.
Hemoglobin is a tetramer comprised of two alpha and two beta subunits. In human Hb, each subunit contains one heme, while the beta (.beta.) subunits also contain highly reactive SH groups (cys.beta.93) (Olson, J. S., Meth. in Enzym., 76:631-651 (1981); Antonini & Brunori, In Hemoglobin and Myoglobin in Their Reactions with Ligands, American Elsevier Publishing Co., Inc., New York, pp. 29-31 (1971)). These cysteine residues are highly conserved among species although their function has remained elusive.
NO (nitric oxide) is a biological "messenger molecule" which decreases blood pressure and inhibits platelet function, among other functions. NO freely diffuses from endothelium to vascular smooth muscle and platelet and across neuronal synapses to evoke biological responses. Under some conditions, reactions of NO with other components present in cells and in body fluids can generate toxic intermediates and products at local concentrations in tissues which are effective at inhibiting the growth of infectious organisms. Thus, it can be seen that a method of administering an effective concentration of NO or biologically active forms thereof would be beneficial in certain medical disorders.